Hum. Reprod. Advance Access originally published online on February 17, 2006
Human Reproduction 2006 21(6):1599-1604; doi:10.1093/humrep/del013
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The limited importance of pronuclear scoring of human zygotes
1 The A.R.T. Institute of Washington, Inc. at Walter Reed Army Medical Center, Washington, DC, 2 Northwest Center for Reproductive Sciences, Kirkland, WA, 3 Department of Obstetrics and Gynecology, Walter Reed Army Medical Center, Washington, DC and 4 Tyho-Galileo Research Laboratories and Reprogenetics, West Orange, NJ, USA
5 To whom correspondence should be addressed at: The A.R.T. Institute of Washington, Inc. at Walter Reed Army Medical Center, PO Box 59727, Washington, DC 20012, USA. E-mail: aida.james{at}na.amedd.army.mil
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Abstract |
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BACKGROUND: Several studies have shown a correlation between the pronuclear morphology score (PNMS) and subsequent embryo development and implantation. Embryos with poor pronuclear score, elsewhere referred to as Z3 and Z4, are often not transferred or cryopreserved because it is believed that they have poor pregnancy potential. The objective of this study is to report our data on the use of the pronuclear score and its effect on pregnancy outcome. METHODS: Retrospective analysis of IVF/ICSI-embryo transfer cycles completed over the course of 1 year (n = 334). Comparisons were made only in those groups of patients in whom cohorts of similarly scored PNMS embryos were transferred. The proportion of such homologous cohorts was 104/334 (31%). All other replacements were excluded from final analysis as they were dissimilar as far as PNMS is concerned. Pregnancy outcomes were evaluated. RESULTS: The incidence of live birth resulting from the transfer of single pronuclear score homologous embryo types was 56 (14/25), 41 (13/32), 54 (23/43) and 0% (0/4) for PNMS scores 1, 2, 3 and 4, respectively. There was no correlation between PNMS category of the embryos transferred and live birth rates (P = 0.139). CONCLUSIONS: PNMSs of 1, 2 or 3 do not correlate with live birth rates when assessing unique PNMS embryo transfers. In particular, previously considered poor (type 3) embryos can result in pregnancy with normal live birth rates. Whether type 4 embryos are compatible with normal development remains to be shown.
Key words: embryo/live birth/pronuclear morphology scores/Z score/zygote
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Introduction |
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Precise selection of embryos and prediction of implantation is probably the most pressing issue in assisted reproduction. There are now a wide variety of methods in place that allow selection. More complicated proposals range from assessing aneuploidy in embryos following biopsy to ways of assessing metabolic turnover rate using a system of assaying culture droplets (Munne et al., 2003
; Brison et al., 2004; Verlinsky et al., 2004; Sher et al., 2005). These systems, although elegant and scientifically fundamental, are not widely applied because they are not straightforward clinically or cost-effective.
More commonly, embryos have been selected based on morphological criteria before and after fertilization. Of particular interest has been the study of phenomena related to development rate, pronuclear and nucleolar behaviour, blastomere fragmentation and multinucleation (Tesarik et al., 1987; Wright et al., 1990; Winston et al., 1991; Munne and Cohen, 1993; Ziebe et al., 1997; Jackson et al., 1998; Alikani et al., 1999; Van Royen et al., 1999, 2001; Scott et al., 2000; Lundin et al., 2001; Montag and van der Ven, 2001; Wharf, 2003).
Phenomena related to pronuclear and nucleolar movements were first described by Wright et al. (1990). Recently, these notions have been expressed in more distinct pronuclear scores and used as a means to select embryos. The scores have been correlated with improved embryo development (Scott et al., 2000; Balaban et al., 2001; Rienzi et al., 2002; Zollner et al., 2002, 2003; Scott, 2003a) as well as with increased pregnancy and implantation (Payne et al., 1997; Scott and Smith, 1998; Ludwig et al., 2000a,b; Scott et al., 2000; Tesarik et al., 2000; Wittemer et al., 2000; Balaban et al., 2001; Zollner et al., 2002, 2003; Scott, 2003a,b; Jaroudi et al., 2004).
Many different pronuclear scoring systems have been proposed to select high-quality embryos. Unfortunately, there is no standard zygote-grading system used throughout assisted reproduction techniques laboratories. The same holds true for other systems of evaluating embryo morphology. As a result, comparisons of the systems used and correlations of embryo quality with success rates between different laboratories are vague. Two main systems for assessing pronuclear morphology were developed by Scott and Smith (1998) and Tesarik et al. (2000). A pronuclear scoring system, as well as other embryo development markers and patient status, may be useful in determining the number of embryos to transfer. As a result, pregnancy and implantation rates may increase, whereas the numbers of high-order multiples would decrease.
The objective of this article was to examine the use of pronuclear scoring in relation to the outcome of live births via the widely used system proposed by Scott and Smith (1998). To increase objectivity, only embryo transfers in which cohorts of embryos with similar pronuclear scores were considered (n = 104), and all other embryo transfers comprised of cohorts of embryos with dissimilar scores were excluded (n = 230).
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Materials and methods |
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This retrospective analysis was approved by the Department of Clinical Investigation at The Walter Reed Army Medical Center in Washington, DC.
Ovarian stimulation and oocyte retrieval
Ovarian stimulation was accomplished following protocols used by the Walter Reed Army Medical Center ART program as determined by age, FSH level and previous ovarian stimulation history. All women were pretreated with oral contraceptive pills (OCP) for a minimum of 14 days before stimulation. Down-regulation consisted of either a long luteal leuprolide acetate protocol (Lupron, TAP Pharmaceuticals, Lakeforest, IL, USA) or a Lupron microdose flare protocol using 40 µg twice daily (Leondires et al., 1999). Ovarian stimulation was accomplished using FSH and/or HMG (Fertinex, Serono, Rockland, MA, USA; Humegon, Organon, Roseland, NJ, USA; or Repronex, Ferring, Suffern, NY, USA) protocol using three to six ampoules of FSH and/or HMG per day beginning on day 3. Oocyte maturation was completed with either 5000 or 10 000 IU of HCG (Profasi, Serono) when the lead follicles measured 
17 mm and/or the estradiol concentration reached 2000–4000 pg/ml.
Oocytes were retrieved via transvaginal ultrasound guidance 35 h after HCG injection. Follicular aspirates were placed directly into isolette chambers (Hoffman Surgical Equipment, Conshohocken, PA, USA) with a warmed, humidified and gassed (37°C, 50–60% relative humidity and 5.5% CO2) environment. Oocytes were isolated and moved to clean microdrops (100 µl of P1 medium, Irvine Scientific, Santa Ana, CA, USA plus 5% human serum albumin (HSA) (100mg/ml) in normal saline, InVitro Care, MD, USA) under mineral oil (M-3516, Sigma Chemicals, St. Louis, MO, USA). Oocytes were then moved to the IVF laboratory and placed in incubators with the same air environment as the isolettes until further use.
Oocyte insemination and embryo culture
Oocyte insemination was initiated 40–41 h after HCG injection using standard IVF and/or ICSI procedures. At 16–18 h after insemination, the presumptive zygotes were assessed for fertilization status. The presumptive zygotes were moved from their fertilization microdrops and placed into clean, individual P1 microdrops under oil and then subsequently assessed for the presence and location of pronuclei and the alignment of nucleolar precursor bodies (NPB) by viewing through different focal planes, concomitantly with assignment of a pronuclear morphological score. The microscopic analysis was completed using an IX70 inverted microscope with Hoffman modulation contrast at x200 magnification (Olympus, Melville, NY, USA).
The scoring system used to assess the zygotes was first developed by Scott et al. (2000) and later refined by Scott (2003a) (Figure 1). Zygotes with two pronuclei were assessed for location and size of pronuclei as well as size, number and distribution pattern of the nucleoli within the pronuclei. The pronuclear morphology scores (PNMSs) of 1 through 4 were allotted according to the following standards. For a score of 1 (also referred to as Z1 in previous literature), the pronuclear oocyte must have equal numbers of nucleoli that are aligned at the furrow between the pronuclei. PNMS 2 (Z2) is assigned to oocytes with equal numbers of nucleoli that are not aligned at the furrow. All other pronuclear oocytes were assigned a score of 3 (Z3) if there were marked differences in size and/or number of nucleoli and the nucleoli were not aligned. The last group of score 4 (Z4) was reserved for the pronuclear oocytes that had different size pronuclei, non-central pronuclei or pronuclei that were not in contact with each other (Sadowy et al., 1998; Scott et al., 2000; Scott, 2003a).
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Embryos were subsequently assessed on a daily basis at 24-h intervals from fertilization checks. At each assessment, embryos were checked for number and evenness of blastomeres, the percentage of fragmentation and nuclear status of each blastomere. On the second day following fertilization check (approximately 80 h after HCG), factors such as the number and quality of embryos, the female patients’ age and the number of previous IVF attempts were used to determine the day of transfer. Those embryos intended for a day 5 transfer instead of a day 3 transfer were placed into extended culture at approximately 100 h after HCG. Extended culture consisted of individual microdrops of CCMTM-30 under oil (VitroLife, Kungsbacka, Sweden). Again, embryos were assessed on a daily basis at 24-h intervals.
On day 3 of in vitro culture, the embryos were assessed for blastomere number, uniformity and degree of fragmentation. The embryos were then assigned a grade according to the criteria developed by Tesarik et al. (2000) and further modified by this laboratory. Grade 1 was assigned to an 8- to 10-cell embryo with no fragmentation. An 8- to 10-cell embryo with <10% fragmentation was given a grade 2. A grade 3 was assigned to either 6- to 8-cell embryos with no fragmentation or 8-cell embryo with 10–20% fragmentation. The embryo grade of 4 was given to any cleavage stage embryo with >20 to <30% fragmentation. Finally, grade 5 was assigned to any embryo that was either arrested or had >30% fragmentation.
Embryo transfer
On the day of embryo transfer (2, 3 or 5), embryos with the best morphology were chosen for transfer regardless of previously assigned pronuclear score. This score, as well as other morphological assessments, was used only in cases where multiple embryos were similarly eligible for transfer. Only a few cases underwent an embryo transfer on day 2 (n = 5), resulting from other circumstances such as poor fertilization. Serum 
HCG was checked on day 14 after oocyte retrieval. If positive, it was repeated in 48 h. An ultrasound was performed approximately 21 days later, and follow-up ultrasounds were performed as indicated clinically. Pregnancies were classified as positive chemical pregnancies if the serum HCG concentrations doubled from day 14 to day 16. They were classified as clinical pregnancies if the presence of a gestational sac with fetal heart tones (FHT) was noted during an ultrasound examination at 6–8 weeks.
Statistical analysis
A retrospective analysis of the pronuclear score associated with all of the embryos transferred to patients (n = 334) at the Walter Reed Army Medical Center IVF program over a 12-month period was completed. This analysis included the final outcomes of positive chemical and clinical pregnancy rates as well as live births associated with these transfers. To determine the true effect of pronuclear scoring on those outcomes, the analysis was narrowed to include only those cases in which one pronuclear score type was transferred (n = 104). All other embryo transfer cases, in which multiple embryos with differing pronuclear scores were replaced, were excluded from analysis because the mixture of embryos that were transferred presented a very complex dataset that would preclude making any conclusions about pregnancy chances from any particular Z score. Therefore, these patients are a subset of the entire clinical population from the same time period.
The positive chemical pregnancy rate, clinical pregnancy rate and live birth outcome variables were treated as binary variables, and the effects of maternal age, day of embryo transfer and the pronuclear score were modelled. Maternal age, day of transfer and the pronuclear score were treated as categorical variables in the model. These categorical distributions were tested with a Mantel–Haenzel test for general association (SAS, 1996). In this outcome analysis, those variables (maternal age and day of transfer) that were not significant (P > 0.05) were removed.
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Results |
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Over a 12-month period that included 334 patients (mean age of 33.4 years), 6092 oocytes were collected, of which 4777 (78%) were mature. The average number of oocytes collected per patient was 18.2. Of the 4777 mature oocytes, 3333 (69.8%) showed normal fertilization (2PN), and the average number of embryos replaced per transfer was 2.9 (Table IA). A total of 641 (19.2%) fertilized zygotes were given a PNMS of 1. A PNMS of 2 was assigned to 934 (28.0%) zygotes, 1427 (42.8%) were given a PNMS of 3 and 331 (9.9%) were assigned a score of 4 (Table IB).
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On day 3, the embryos were graded on morphological parameters. Of the 3287 embryos that developed to day 3 of culture, 303 (9.2%) were grade 1. Morphology grade 2 was assigned to 672 (20.4%) embryos. A total of 955 (29.1%) embryos exhibited morphological characteristics consistent with a grade 3. Finally, grades 4 and 5 were assigned to 756 (22.9%) and 601 (18.3%) of the remaining day 3 embryos, respectively (Table IC).
Overall, 912 embryos were transferred into 334 patients during the time period set for the retrospective analysis. The clinical pregnancy rate for the entire population was 47% and the live birth rate was 38% as reported to Society of Assisted Reproductive Technologies (http://www.cdc.gov/reproductivehealth/ART/index.htm). The overall rate of multiples was 32% (50/155) of all live birth events, with a high-order multiple rate of 4% (6/155). For the retrospective analysis comparing similar PNM-scored zygotes, 234 embryos were transferred into 104 patients. The multiple live birth rate for the retrospective analysis was 24% (12/50), with a high-order multiple rate of 4% (2/50).
Twenty-five patients received only PNMS 1 embryos (total = 54 embryos, range = 1–4 embryos per patient). Nineteen gestational sacs were visualized, giving an implantation rate of 35.2% (19/54) for PNMS 1 transfers. For those patients (n = 32) receiving only PNMS 2 zygotes, 69 embryos were replaced (range = 1–4 embryos per patient). An implantation rate of 23.2% was calculated for PNMS 2 transfers after 16 gestational sacs were detected. In the PNMS 3 category, 43 patients received 106 embryos (range = 1–4 embryos transferred per patient). Upon ultrasound examination, the PNMS 3 transfer patients had 36 gestational sacs to give an implantation rate of 34.0%. The remaining four patients had five PNMS 4 embryos replaced, with a range of 1–2 embryos per transfer. There were 0 gestational sacs for this transfer group (Table ID).
Although maternal age was independently correlated with pregnancy outcomes, age was not predictive of PNMS. Likewise, PNMS was not predictive of live birth, clinical pregnancy or positive chemical pregnancy (P = 0.092, 0.392, 0.416 and 0.679 for scores 1, 2, 3 and 4, respectively) (Table II).
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The positive chemical pregnancy rate, established by HCG concentration, for all of the embryo transfer groups that consisted of a single PNMS type was 56% (58/104). From 25 patients who received PNMS 1 embryos, 15 (60%) had positive HCGs. Thirty-two patients in the PNMS 2 embryos transfer group had 16 (50%) positive HCGs. In the PNMS 3 embryo transfer group of 43 patients, the number of positive HCGs was 27 (63%). Transfer of embryos with a single PNMS of 4 resulted in 0 pregnancies; however the number (n = 4) of score 4 patients was too small to be conclusive (Table II).
The overall clinical pregnancy rate for the analysed transfer groups was 50% (52/104). Of the 25 patients who received an embryo transfer of PNMS 1 embryos, 15 (60%) had positive FHTs. In the PNMS 2 embryo transfer group of 32 patients, 13 (41%) had positive FHTs. For the 43 patients in the PNMS 3 embryo transfer group, the number of positive FHTs was 24 (56%) (Table II).
Overall, live birth rate for the analysed transfer groups was 48% (50/104). The number of live births that resulted from the transfer of embryos with a single PNMS ranged from 0 (score 4) to 56% (score 1); however, as with HCG and FHT outcomes, the number of score 4 patients (n = 4) was too small to be conclusive. From 25 patients who received PNMS 1 embryos, 14 (56%) delivered healthy babies. Of 32 patients who received only PNMS 2 embryos, 13 (40%) delivered liveborn. When only PNMS 3 embryos were transferred to 43 patients, the number of live births was 23 (54%) (Table II). The categorical distributions of live birth associated with PNMS resulted in a P-value of 0.139. No association was found between the PNMS of the embryos transferred and the outcome of live birth. Live birth rate in the PNMS score (Z3) was entirely normal, despite low anticipated prognosis.
There are differences in pregnancy rates between day 3 and day 5 transfers within each PNMS group but not between PNMS groups, which is in agreement with current thoughts on day 3 versus day 5 transfers (Scholtes and Zeilmaker, 1996; Gardner et al., 1998, 2000; Marek et al., 1999; Coskun et al., 2000; Huisman et al., 2000; Milki et al., 2000) (Table III).
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Discussion |
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This study showed no differences in positive chemical pregnancy rates, clinical pregnancy rates or live birth rates when transfers on either day 3 or day 5 were analysed by the four different PNMSs (1, 2, 3 and 4). These data provide evidence that embryos in pronuclear morphology categories 1, 2 and 3 have similar viability. This is in contrast with the data reported by a number of other studies that claim there is zero chance that the category 3 embryos will produce a live birth. Tesarik and Greco (1999)
reported a 50% (22/44) pregnancy rate when at least one high-grade embryo (pattern 0) was transferred versus a 9% (2/23) rate for transfers with embryos derived from abnormal zygotes (patterns 1–5). One study concluded that pattern 0 zygotes produced more good-quality embryos with greater implantation potential than embryos from abnormal zygote patterns (Wittemer et al., 2000). Pattern 0 embryos (Tesarik and Greco, 1999) correlate to PNMSs 1 and 2 as developed by Scott and Smith (1998). Scott et al. (2000) reported clinical pregnancies for day 3 transfers only when embryos with PNMS of 1 (Z1) or 2 (Z2) were included in the transfer. In another study, Scott (2002) reported no live offspring when embryos were transferred exclusively from zygotes that had a PNMS of 3 (Z3) or 4 (Z4). It is possible that the dataset in those studies were biased by a skewed distribution. Unlike Tesarik and Greco (1999), Scott et al. (2000) or Wittemer et al. (2000), the current study shows an equal chance of pregnancy and live births when there were only PNMS 3-derived embryos transferred on day 3 (Table III). Moreover, Jaroudi et al. (2004) showed no correlation between selecting higher quality zygotes and pregnancy rates when embryos were selected for transfer according to the system of Tesarik and Greco (1999), as well as the revised Z-scoring system of Scott et al. (2000). Salumets et al. (2001) also reported no difference in pregnancy and implantation rates between pattern 0 and non-pattern 0 zygotes in single-embryo transfer patients. Montag and van der Ven (2001) reported on a multi-centre study in a country (Germany) that has legislated embryo transfer limits that would require embryo selection before development beyond the pronuclear stage. When embryo transfers were performed based on pronuclear morphology, the study reported pregnancy and implantation rates that showed some differences between the groups. Despite the differences, the study did show that pregnancy and implantation did occur even when the pronuclear morphology was not the equivalent to PNMS 1 or 2.
Balaban et al. (2001) reported that embryos with an ideal pronuclear pattern were more likely to develop into high-grade blastocysts as well as attain higher implantation and pregnancy rates compared with those embryos from a non-ideal pronuclear pattern. They also found that blastocysts from abnormal pronuclear patterns would produce pregnancies but at a lower rate. Scott et al. (2000) reported that when only PNMS 3-derived blastocysts were available for transfer on day 5, there were no pregnancies (n = 6). In contrast, when only PNMS 3-derived blastocysts were transferred in the current study, the live birth rate was 100% (n = 4) (Table III).
Embryos with a PNMS of 4 are fairly uncommon, occurring <10% of the time in this study; the transfer set of uniquely score 4 embryos is even rarer (1%, 4/334). When analysed by single-score transfer, none of the four patients with a PNMS of 4 became pregnant. It is important to emphasize that those conclusions drawn from small sample sizes of biological comparisons can both exaggerate or obscure an observation.
Assessing a dynamic event such as pronuclear morphology is extremely difficult to do in a clinical setting because of the extreme importance of timing. Thus far, laboratories have been assessing this trait using a time range from the beginning of an insemination procedure. There are a few reasons for using a time range, one of which is the limited time range in which pronuclei can actually be visualized for analysis. Another reason to use a time range is the difference in time of fertilization that depends upon the method of insemination. Technicians know when certain oocytes have been fertilized in ICSI cases because the sperm is individually injected into each oocyte. However, it is only a best guess as to when the oocytes of an IVF case were fertilized. In some laboratories, the oocytes and sperm are together for as short a time period as 4 h and in the others this period may be extended to 18 h. There is no way of knowing which oocytes fertilized at which time point within that fertilization time period. Montag and van der Ven (2001) showed a difference in the availability of higher PNM-scored embryos when comparing ICSI versus IVF embryos. This most likely correlates to the difference in the timing of insemination between the two treatment groups.
Pronuclear development is not a static event (Tesarik and Kopecny, 1989; Payne et al., 1997; Tesarik and Greco, 1999; Scott, 2003b); therefore, disregarding an embryo based on a single time assessment can be misleading. Zygotes that are assigned a poor pronuclear score may in fact become high-grade zygotes when assessed at a later time. Sadowy et al. (1998) who analysed chaotic mosaicism in zygotes with uneven pronuclei never indicated that all such zygotes were abnormal. Also, the analysis was conducted with biometric evaluation, a system of evaluation that has not been repeated in any of the subsequent pronuclear studies. Nevertheless, Scott (2002) considers poor pronuclear score zygotes as being unfit for transfer or freezing. Wright et al. (1990) and Demirel et al. (2001) reported that early in pronuclear development, the nucleoli are small, numerous and distributed in a random pattern, but that as time proceeds, the nucleoli will coalesce and begin to align near the furrow between the pronuclei. Therefore, timing can have a drastic effect on the assessment of embryo quality when static observations are used (Payne et al., 1997; Demirel et al., 2001; Montag and van der Ven, 2001; Jaroudi et al., 2004). Jaroudi et al. (2004) suggested that when used as the only selection criteria, zygote scoring might not be sufficient to select the most viable embryos. Thus, a single observation may not reveal the true zygote morphology because it cannot be determined if nuclear development is in progress or has never proceeded when syngamy is initiated.
The difference observed in live birth rates between embryos transferred on day 3 (46%) and day 5 (73%) is not an unexpected result in this study. The day of embryo transfer (day 3 versus day 5) is determined using a strict set of selection criteria (number of previous IVF attempts, maternal age, number of transferable embryos and morphology on day 3 of development). By meeting these strict selection criteria, embryos allowed to remain in culture until day 5 are inherently of better quality on day 3 of culture than larger sets of embryos that do not meet the strict selection criteria and need to be transferred on day 3 (Behr et al., 1999).
In conclusion, we found that PNMS 3-derived embryos are just as likely as PNMS 1 or PNMS 2 embryos to produce a pregnancy that results in a child. Although in the past it has been reported that PNMS 3 embryos will not lead to a live birth (Scott, 2002), here we have found that pregnancy and ultimately live birth can be achieved by transferring PNMS 3-type embryos. This is important because many laboratories may be relying on previously reported data that PNMS 3 embryos do not produce successful ongoing pregnancies and therefore may be discarding embryos with real potential.
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The authors thank Kerry Polson, Donna Hoover, Darshana Naik and Stephanie Hosid for their contributions to data collection, as well as Jim Segars and Adam Hamm for their technical assistance.
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References |
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Submitted on April 24, 2005; resubmitted on December 1, 2005; accepted on January 10, 2006.
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